Trametinib sensitizes KRAS-mutant lung adenocarcinoma tumors to PD-1/PD-L1 axis blockade via Id1 downregulation

Background The identification of novel therapeutic strategies to overcome resistance to the MEK inhibitor trametinib in mutant KRAS lung adenocarcinoma (LUAD) is a challenge. This study analyzes the effects of trametinib on Id1 protein, a key factor involved in the KRAS oncogenic pathway, and investigates the role of Id1 in the acquired resistance to trametinib as well as the synergistic anticancer effect of trametinib combined with immunotherapy in KRAS-mutant LUAD. Methods We evaluated the effects of trametinib on KRAS-mutant LUAD by Western blot, RNA-seq and different syngeneic mouse models. Genetic modulation of Id1 expression was performed in KRAS-mutant LUAD cells by lentiviral or retroviral transductions of specific vectors. Cell viability was assessed by cell proliferation and colony formation assays. PD-L1 expression and apoptosis were measured by flow cytometry. The anti-tumor efficacy of the combined treatment with trametinib and PD-1 blockade was investigated in KRAS-mutant LUAD mouse models, and the effects on the tumor immune infiltrate were analyzed by flow cytometry and immunohistochemistry. Results We found that trametinib activates the proteasome-ubiquitin system to downregulate Id1 in KRAS-mutant LUAD tumors. Moreover, we found that Id1 plays a major role in the acquired resistance to trametinib treatment in KRAS-mutant LUAD cells. Using two preclinical syngeneic KRAS-mutant LUAD mouse models, we found that trametinib synergizes with PD-1/PD-L1 blockade to hamper lung cancer progression and increase survival. This anti-tumor activity depended on trametinib-mediated Id1 reduction and was associated with a less immunosuppressive tumor microenvironment and increased PD-L1 expression on tumor cells. Conclusions Our data demonstrate that Id1 expression is involved in the resistance to trametinib and in the synergistic effect of trametinib with anti-PD-1 therapy in KRAS-mutant LUAD tumors. These findings suggest a potential therapeutic approach for immunotherapy-refractory KRAS-mutant lung cancers. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s12943-024-01991-3.


Background
Lung cancer is the leading cause of cancer death worldwide [1].Genetic characterization of oncogenic driver mutations (EGFR, KRAS, ALK, ROS) has transformed the management of non-small cell lung cancer (NSCLC) therapy with the introduction of molecularly targeted therapies, allowing an individualized treatment approach [2,3].KRAS mutations represent the most prevalent genetic driver alteration in lung adenocarcinomas (LUAD) [2].Despite the approval of KRAS G12C inhibitors, adagrasib (MRTX849) and sotorasib (AMG510) for NSCLC patients harboring KRAS G12C mutations, KRAS alterations are still related to unfavorable therapeutic outcomes and improvement in survival rates in this LUAD subtype remains an unmet clinical need [4,5].
Targeting KRAS signaling at the level of its downstream effectors, such as mitogen-activated protein kinases (MAPK), has also been studied [6].Trametinib (GSK1120212) is an oral, reversible and selective allosteric inhibitor of MEK1/2 and is FDA-approved for the treatment of metastatic BRAF V600E mutant NSCLC and melanoma, in combination with BRAF inhibitors [7,8].However, the antitumor activity of MEK1/2 inhibitors in monotherapy or in combination with conventional chemotherapy has been demonstrated to be ineffective in a significant proportion of patients with KRAS-mutant advanced NSCLC [9,10].Therefore, diverse combination therapies employing targeted therapies or monoclonal antibodies, such as programed-cell death protein 1 (PD-1)/ programed-cell death-ligand 1 (PD-L1) inhibitors, are emerging in an attempt to overcome primary drug resistance and/or acquired resistance to trametinib [11,12].
Immune checkpoint inhibitors (ICIs) have shown unprecedented clinical results in advanced NSCLC, providing durable responses and prolonging long-term survival [13].However, only a small subset of KRAS-mutant lung cancer patients obtains a significant long-term benefit from these treatments [14][15][16].These drugs reactivate antigen-specific antitumor effector T cells, boosting the immune response against cancer cells.A range of studies have demonstrated that molecularly targeted therapies can modulate tumor antigenicity and potentiate T cell immune recognition, improving the efficacy of ICIs [17,18].Recently, preclinical studies in KRASmutant murine LUAD models demonstrated the therapeutic benefit of combining inhibitor of differentiation-1 (Id1) blockade with ICIs.Id1 gene silencing enhanced PD-L1 expression on lung cancer cells, sensitizing tumor cells to PD-1/PD-L1 axis blockade and enhancing CD8 + T cell infiltration [19].Id1 is a member of the inhibitor

Graphical Abstract
of differentiation (Id1-Id4) family.Id proteins, which are described as dominant negative transcriptional regulators, are overexpressed in human cancers, including lung malignancies [20,21].In the KRAS-mutant LUAD setting, Id1 expression, known to provide an aggressive pro-oncogenic phenotype and to enhance the colonization capacity of lung cancer cells, has been shown to correlate with reduced overall survival and poor treatment response [22][23][24].
Based on these premises, we aimed to evaluate the effect of trametinib in Id1 in different KRAS-mutant LUAD cells and preclinical in vivo models.Here we provide key evidence on the preclinical and functional role of Id1, sustaining MAPK (KRAS-MEK1/2) signaling in the acquired resistance to trametinib and PD-1/PD-L1 blockade in KRAS-mutant LUAD tumors via PD-L1 downregulation.

Cell lines and reagents
Human LUAD cell lines (H1792, H2009, H23, A549, H2030, H1437, H1568, and H2126) and murine LUAD cell line Lewis Lung Carcinoma (LLC) were purchased from the American Type Culture Collection.KLA LUAD cells were derived from KR181 fl/fl mice [25].393P cells were derived from KRAS LA1/+ ; p53 R172HDG mice, and provided by Dr. J. M. Kurie (The University of Texas, MD Anderson Cancer Center, Houston, TX, USA).Lacun3 LUAD cell line, which was established from a chemically induced LUAD mice model [26], was a gift of Prof. L. Montuenga (Cima Universidad de Navarra, Pamplona, Spain).CMT167 LUAD cell line was kindly provided by Dr. F. Torres Andon (Center for Research in Molecular Medicine and Chronic Diseases, Santiago de Compostela, Spain).Murine and human cells were cultured at 37 °C and 5% CO 2 in DMEM (Gibco, Waltham, MA, USA) or RPMI 1640 medium (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum and 1% Penicillin/Streptomycin (Gibco, Waltham, MA, USA).Cells were routinely tested for Mycoplasma with the MycoAlert Mycoplasma Detection Kit (Lonza, Basel, Switzerland).
Trametinib-resistant (TR) cell lines were generated by culturing murine (CMT167, LLC and KLA) and human (H1792, H2009, A549 and H2030) parental lines with increasing concentrations of trametinib up to 500 nM, as previously described [27].TR cell lines were always cultured in cell media with trametinib (500 nM) which was renewed every 4 days.TR-cell resistance to trametinib was measured by analyzing the proliferation capacity of these cells in the presence of trametinib, and compared with parental cells.
Western blot analysis were performed as previously described [23].The antibodies used and their dilutions are indicated in Supplementary Table S1.The quantification of the Western blot band intensity was done using Image Lab software (Bio-Rad, CA, USA), and normalized with the density of the loading control protein.RNA extraction and real-time PCR were performed as previously described [23].The sequences of the primers used in the real-time PCR assays are indicated in Supplementary Table S2.
For proliferation assays, 2500 cells per well were seeded in 96-well plates in 100 μL of complete medium in triplicate for each experimental condition.Cell proliferation was assessed at 72 h using trametinib concentrations ranging from 2000 nM to 0.008 nM.Cells were fixed with formaldehyde and stained with a commercial 1% crystal violet solution (V5265, Sigma-Aldrich, Saint Louis, MO, USA).A 20% acetic solution was used to dissolve crystal violet, and the absorbance was measured at 590 nm in a SPECTROstar Nano (BGM LABTECH, Ortenberg, Germany).

RNA sequencing
RNA sequencing data analysis was performed using the following workflow: (1) the quality of the raw reads was verified using FastQC software and the trimming of the reads was carried out with trimmomatic [28]; (2) alignment against the mouse reference genome (GRCh38) was performed using STAR [29]; (3) gene expression quantification using read counts of exonic gene regions was carried out with featureCounts [30]; (4) the gene annotation reference was Gencode v42 [31]; and (5) differential expression statistical analysis was performed using R/Bioconductor (https:// www.R-proje ct.org/) as follows.Gene expression data was normalized with edgeR [32] and voom [33].After quality assessment and outlier detection using R/Bioconductor, a filtering process was performed.Genes with read counts lower than 4 in more than the 50% of the samples of all the studied conditions were considered as not expressed.LIMMA was used to identify the genes with significant differential expression between experimental groups.Genes were selected as differentially expressed using a cut-off value of B > 5. Further functional and clustering analyses and graphical representations were performed using the R/Bioconductor and clusterProfiler [34].Data are publicly available in GEO database with the accession number GSE236258.
To assess the antitumor effect of trametinib and its combination anti-PD-1, LLC or 393P (4 × 10 6 ) cells were subcutaneously injected in the right flank of female C57BL/6 J and Sv/129 mice, respectively.LLC and 393P tumors were allowed to grow for 7 and 14 days before treatment, respectively.Tumor-bearing mice were randomized in four groups and treated with anti-PD-1 (100 mg/mouse; intraperitoneally, twice a week; RMP1-14 BioXCell, Lebanon, NH, USA), trametinib (1 mg/kg; oral oral administration, 5 times a week), their combination, or vehicle for three weeks.
Tumors were measured periodically using a digital caliper (DIN862, Ref 112-G, SESA Tools, Hernani, Spain), and tumor volume was calculated using the following formula:

Flow cytometry analyses
Tumors were dissected, cut into small pieces, and incubated with 1 mg/ml collagenase D and 50 µg/ml DNase I (Roche, Basel, Switzerland) at 37 °C for 30 min.EDTA (6 µM) was then used to block collagenase and DNase I activities.Then, tumors were mechanically disaggregated through a 70 μm cell strainer (Corning, NY, USA).Erythrocytes were lysed with ACK buffer (Gibco, Waltham, MA, USA).Single cell suspensions were layered over 35% Percoll (GE Healthcare, IL, USA) and centrifuged to purify the tumor infiltrating leukocytes for flow cytometry analysis.Cell suspensions were incubated with a purified monoclonal antibody against CD16/CD32 (Mouse BD Fc Block, 1:200, BD Bioscience, NJ, USA) for 15 min at 4ºC and stained with fluorochrome-conjugated antibodies diluted in FACS buffer (PBS, 5% Fetalclone, 2.5 mM EDTA).For intracellular staining, cells were permeabilized with eBioscience Fixation/Permeabilization Kit (Invitrogen, Waltham, MA, USA) for 15 min and stained.The antibodies used for flow cytometry analyses are listed in Supplementary Table S3.Dead cells were excluded using PromoFluor-840 NIR Maleimide (Pro-moCell, Heidelberg, Germany).Gating strategies are shown in Supplementary Figs.S1 and S2.
Quantification of apoptosis was assessed by flow cytometry as previously described [19].PD-L1 expression was assessed by flow cytometry, as previously described [2].Briefly, LUAD cells were seeded into 6-well plates.After 24 h, human and murine lung cancer cells were treated with trametinib for 72 h.Trametinib concentrations used were 100 nM for murine and 500 nM for human LUAD cells.Control cells were treated with DMSO.After 48 h of incubation, recombinant murine (500 U/mL, PeproTech, MA, USA) or human (20 ng/ ml, Biolegend,CA, USA) IFN-γ was added, and the cells were incubated for 24 h.Cells without IFN-γ were also analyzed.PE-conjugated anti-CD274 (PD-L1) was used to detect PD-L1 expression on lung cancer cell surface.Cells were acquired using a CytoFLEX LX flow cytometer (Beckman Coulter, CA, USA) and analyzed with CytExpert software (Beckman Coulter, CA, USA).

Statistical analysis
Data normality was assessed using the Shapiro-Wilk test.Parametric comparations between experimental groups were performed by two-sided t-test or one-way ANOVA test followed by the Tukey post hoc test.Non-parametric comparisons between experimental groups were performed by two-sided Mann-Whitney U-test or Kruskal-Wallis test with post hoc Mann-Whitney U-test.Survival curves were generated using the Kaplan-Meier method, and differences were analyzed with the log-rank test.p < 0.05 was considered statistically significant.Statistical analyses were performed using the version 9 of Graph-Pad Prism software (GraphPad Prism 9, San Diego, CA, USA).

Trametinib reduces Id1 levels in NSCLC cells in vitro and in vivo
The constitutive activation of the MAPK pathway as a result of KRAS mutations has led to the use of trametinib as a potential therapeutic option to treat KRAS-mutant cancers [35].We first assessed the cytotoxic effect of trametinib in a panel of murine and human lung cancer cells.A significant impairment of cell growth was observed in all murine and human KRAS-mutant LUAD cell lines (Fig. 1A and B).Nonetheless, proliferation of KRAS-wild type lung cancer cells seemed not to be affected by trametinib (Fig. 1C).Among the KRAS-wild type lung cancer cell lines analyzed, trametinib only impaired the viability of the H1437, a cell line that harbors MAP2K1 mutation [36].These observations suggest that the antitumor activity of trametinib is mainly associated with KRAS mutational status.
Previously, we found that Id1 is a key factor of the MAPK signaling pathway in KRAS-mutant human LUAD [24].Interestingly, trametinib treatment diminished Id1 protein levels in KRAS-mutant and KRAS-wildtype LUAD cells.The magnitude of this downregulation was even higher than the observed with a short hairpin (sh) RNA targeting Id1 (sh-Id1) in most of cell lines studied (Fig. 1D and E).
Overall, these data suggest that trametinib reduces Id1 levels in vitro and in vivo in KRAS-mutant LUAD, and impairs lung cancer tumor growth.

Trametinib enhances proteasome activity to degrade Id1
In order to investigate the molecular mechanisms involved in the trametinib-mediated Id1 protein downregulation, we first analyzed by real-time PCR the effects of trametinib on Id1 mRNA relative expression in a panel of mouse and human trametinib-treated KRAS-mutant LUAD cells.In contrast to the effects observed for Id1 protein levels, an increase in Id1 mRNA levels were observed in most KRAS-mutant trametinib-treated cells (Fig. 2A).This suggests that trametinib treatment is associated with a post-translational downregulation of Id1 protein.
A transcriptomic profiling using RNA sequencing (RNA-seq) data was performed on murine CMT167 and KLA lung cancer cells treated or not with trametinib.Functional analyses (GO) identified a significant enrichment of pathways related with the proteasome-ubiquitin system in trametinib-treated cells compared with control cells (Fig. 2B).The proteasome system regulates intracellular protein degradation, so it may be involved in trametinib-mediated Id1 inhibition [37].Remarkably, proteasome blockade using a specific inhibitor (MG-132) abolished trametinib-mediated Id1 downregulation in mouse as well as in human KRAS-mutant LUAD cells (Fig. 2C).Of note, CQ, an autophagy inhibitor, did not affect trametinib-mediated Id1 inhibition in trametinibtreated mouse and human KRAS-mutant LUAD cells [38], suggesting that autophagy was not involved in trametinib-mediated Id1 downregulation (Supplementary Fig. S4A).
Previous studies demonstrate that E3 ubiquitin ligases mediate ubiquitination and degradation of Id family members [39,21].RNA-seq analysis on murine CMT167 and KLA KRAS-mutant LUAD cells treated or not with trametinib demonstrated that trametinib upregulates the expression of SMURF-2 and FBXW7 E3 ubiquitin ligases (Supplementary Fig. S4B).These data were validated by real-time PCR and Western blot analysis in human as well as in mouse KRAS-mutant LUAD cells (Fig. 2D  and E).Interestingly, sh-mediated silencing of SMURF2 restored Id1 protein levels in trametinib-treated human as well as murine KRAS-mutant LUAD cells (Fig. 2F).This result suggests that trametinib-mediated Id1 downregulation is induced, at least in part, by SMURF2-mediated proteasome-dependent degradation.

Id1 is associated with the acquired resistance to trametinib in KRAS-mutant NSCLC cells
The failure of trametinib in clinical trials performed in patients with KRAS-mutant NSCLC has been determined mainly by the appearance of acquired resistance [9].The identification of the biological mechanisms implicated in resistance to MEK inhibition has been stablished as a strategy to overcome the resistance to these inhibitors [40].To analyze such mechanisms, murine (CMT167, LLC and KLA) and human (H1792, H2009, A549 and H2030) trametinib resistant (TR) KRAS-mutant LUAD cells were generated as previously described [27] and tumor cells were considered TR when their trametinib IC50 at 72 h became significantly higher than the IC50 of parental cells treated with trametinib [41] (Supplementary Fig. S5A).
Reactivation of the MAPK pathway has been reported as a mechanism of acquired resistance to MEK1/2 inhibition [40].In view that Id1 is a key factor involved in this pathway [24], we studied the effects of trametinib on the Id1 protein expression levels in murine and human TR KRAS-mutant LUAD cells.In contrast to the effect observed in parental cell lines, Id1 protein levels were not reduced in TR cells (Fig. 3A).Interestingly, the RNA-seq analysis of parental CMT167 cells and TR CMT167 cells indicated a downregulation of SMURF2 and upregulation of Id1 mRNA in TR cells when compared with their parental-treated counterparts (Supplementary Fig. S5B).Next, we explored the potential contribution of Id1 to the acquired resistance to trametinib-mediated apoptosis, a core mechanism of trametinib-mediated cancer cell death [42,43], in TR KRAS-mutant mouse and human LUAD cells.In the presence of trametinib, the proportion of apoptotic cells was significantly lower in TR cells when compared to their parental counterparts in all the KRAS-mutant mouse and human LUAD cell lines analyzed.Importantly, the inhibition of Id1 by specific shRNAs (sh1-Id1 and sh2-Id1 in mice and sh-Id1 in human) (Supplementary Fig. S5C) in TR cells restored the levels of trametinib-mediated apoptosis (Fig. 3B).
To evaluate the functional implication of Id1 in trametinib acquired resistance, exogenous Id1 (Id1flag) was expressed in KRAS-mutant mouse (CMT167, LLC and 393P) and human (H1792 and H2009) LUAD cells.Overall, the magnitude of the trametinib-mediated Id1 reduction was markedly lower in cell lines with Id1-flag expression when compared with their GFP-expressing counterparts (Control) (Fig. 3C; Supplementary Fig. S5D).Importantly, Id1-flag expression rendered KRAS-mutant mouse and human LUAD cells resistant to trametinib treatment (Fig. 3C; Supplementary Fig. S5D), suggesting a role of Id1 in the acquired resistance to trametinib treatment.Consistently, the inhibition of Id1 expression by a lentiviral shRNA (sh-Id1) significantly reduced the viability of TR KRASmutant human LUAD cells.This viability was rescued when the levels of Id1 were restored by retroviral transduction of Id1 cDNA refractory to sh-Id1 (Id1-OE) in TR KRAS-mutant human LUAD sh-Id1 cells (Fig. 3D).Moreover, the rescue of Id1 levels in human TR KRASmutant LUAD sh-Id1 cells also enhanced the protein levels of FOSL1 and cMyc in the absence of ERK1/2 activation (Fig. 3E), which are associated with the KRAS oncogenic pathway and/or have been associated with trametinib resistance [24,44,45].
Overall, these data suggest a prominent role of Id1 in the acquired resistance of LUAD cells to trametinib treatment.
(See figure on next page.)Fig. 2 Trametinib increases proteasome activity to reduce Id1 levels in KRAS-mutant LUAD cells.A Id1 mRNA expression levels assessed by real-time PCR in KRAS-mutant mouse (upper panel) and human (lower panel) LUAD cells treated with trametinib (TRAM) (100 nM in murine cells and 500 nM in human cells) for 72 h or vehicle (Control).β-ACTIN was used as the reference gene.B GO Biological process (BP) enrichment analysis of murine KRAS-mutant LUAD cells (CMT167 and KLA) treated with trametinib (TRAM) (100 nM) for 72 h or vehicle (Control).C Western blot analysis of Id1 protein levels in KRAS-mutant mouse (left panel) and human (right panel) LUAD cell lines treated with trametinib (TRAM) (100 nM in murine cells and 500 nM in human cells) for 72 h or vehicle (Control), in the presence or not of the proteasome inhibitor MG-132 (5 μM; 6 h).ß-actin and HSP90 were used as controls for mouse and human cell lines, respectively.D E3 ubiquitin ligases SMURF2 and FBXW7 mRNA expression levels assessed by real-time PCR in KRAS-mutant mouse (upper panel) and human (lower panel) LUAD cells treated with (TRAM) (100 nM in murine cells and 500 nM in human cells) for 6 h or vehicle (Control).β-ACTIN was used as the reference gene.E Western blot analysis of the E3 ubiquitin ligases SMURF2 and FBXW7 in KRAS-mutant mouse (upper panel) and human (lower panel) LUAD cell lines treated with trametinib (TRAM) (100 nM in murine cells and 500 nM in human cells) for 6 h or vehicle (Control).β-actin was used as control.F Western blot analysis of the E3 ubiquitin ligase SMURF2 and Id1 in KRAS-mutant mouse (left panel) and human (right panel) LUAD cell transduced with lentiviral shRNAs targeting SMURF2 or with a scrambled sequence (Control).Cells were treated with trametinib (TRAM) (100 nM in murine cells and 500 nM in human cells) for 72 h or vehicle (Control).GAPDH and β-actin were used as control.In Western blot analyses, relative optical density is indicated underneath each lane.Data are expressed as mean ± SD.Comparisons between experimental groups were performed by two-sided t-test (parametric) or Mann-Whitney U-test (non-parametric)
Next, we characterized by IHC the changes in LLC tumor-infiltrating immune cell populations resulted from trametinib and anti-PD-1 combined treatment.Trametinib and anti-PD-1 combined treatment led to a significant increase in tumor-infiltrating CD3 + T cells and CD8 + T cells whereas no differences in the frequencies of tumor-infiltrating CD4 + T cells were observed.Moreover, the frequency of tumor-infiltrating Foxp3 + cells (Treg cells) were significantly reduced in mice treated with trametinib and anti-PD-1 combination.Interestingly, the CD8 + /Treg cell ratio was significantly increased in the combined treatment group.Of note, as shown in Fig. 1F Id1 expression in tumor tissues was significantly reduced in trametinib-treated mice (Fig. 6A).
We further characterized by flow cytometry the changes in the immune populations resulting from the combined treatment.The experiment was performed 14 days after LLC tumor inoculation (Supplementary Fig. S8A).As observed before, the combined treatment led to an increase of tumor-infiltrating CD8 + T cells, a reduction in the proportion of Treg cells and a significant increase of the CD8 + /Treg cell ratio (Fig. 6B; Supplementary Fig. S8B).Besides, an increase in B cells was observed in the combined treatment (Supplementary Fig. 3 Id1 is involved in the acquired resistance of KRAS-mutant LUAD cells to trametinib treatment.A Western blot analysis of Id1 protein in parental and trametinib resistant (TR) KRAS-mutant mouse (left panel) and human (right panel) cells lines.Cells treated with trametinib (TRAM) (100 nM in murine cells and 500 nM in human cells) for 72 h or vehicle (Control).ß-actin and HSP90 were used as controls for mouse and human cell lines, respectively.B Flow cytometry analysis of the proportion of apoptotic parental and TR KRAS-mutant mouse (upper panel) and human (lower panel) cells.Parental cells were treated with trametinib (TRAM) (100 nM in murine cells and 500 nM in human cells) for 72 h or vehicle (Control).Id1 was silenced in TR cells using lentiviral transduced shRNAs (sh1-Id1 and sh2-Id1 in mice and sh-Id1 in human) or with a scrambled sequence (Control) TR-cell lines were cultured in cell media with trametinib (500 nM).Apoptosis was assessed by flow cytometry by Annexin V and 7AAD staining, as previously described [19,24].C Upper panel: Western blot analysis of Id1 (17 kDa) and Id1-flag (20 kDa) proteins.Control cells were transfected with a GFP cDNA expressing vector (control).Cells were treated with trametinib (TRAM) (100 nM in murine cells and 500 nM in human cells) for 72 h or vehicle (Control).β-actin was used as control.Lower panel: Effects of exogenous Id1 (Id1-flag)-transduction in the survival of KRAS-mutant mouse (CMT167, LLC) and KRAS-mutant human (H1792 and H2009) trametinib-treated tumor cells.Trametinib IC50 is indicated in the figure.D Upper panel: Western blot analysis of Id1 protein in TR KRAS-mutant human cells lines.GAPDH was used as control.Id1 was silenced using the sh1-Id1.The expression of Id1 in sh1-Id1-transduced cells was rescued by retroviral transduction of Id1 cDNA refractory to sh-Id1 inhibition (sh1-Id1/Id1-OE) [24].Cells treated with Trametinib (TRAM) 500 nM for 72 h or vehicle (Control).Lower panel: Cell viability of the upper panel cells when treated with trametinib (TRAM) 500 nM for 72 h or vehicle (Control).E Western blot analysis of c-Myc, total and phosphorylated p42 MAPK, total and phosphorylated p44 MAPK and FOSL1 in parental and in TR KRAS-mutant human cells lines treated as shown in the upper panel of D. GAPDH was used as control.In Western blot analyses, relative optical density is indicated underneath each lane.Data are expressed as mean ± SD.Comparisons between experimental groups were performed by one-way ANOVA followed by the Tukey post hoc test  or a GFP cDNA expressing vector (Control).Cells were treated with trametinib (TRAM) (100 nM in murine cells and 500 nM in human cells) for 72 h or vehicle (Control).Mouse cells were incubated or not with murine IFN-γ (1500 U/ml) for 24 h and human cells were incubated or not with human IFN-γ (20 ng/ml) for 24 h.Data are expressed as mean ± SD.Comparisons between experimental groups were performed by one-way ANOVA test followed by the Tukey post hoc test and polymorphonuclear (PMN) myeloid-derived suppressor cells (MDSCs).No remarkable differences were observed for the proportions of monocytic MDSCs (MON-MDSCs) (Fig. 6B).A significant increase of tumor-infiltrating DCs and M1 macrophages was observed in the combined group (Fig. 6B and Supplementary Fig. S8B).No remarkable differences were observed in the proportions of tumor-infiltrating total and M2 macrophages (Supplementary Fig. S8B).
Taken together, these findings indicate that trametinib reduces Id1 levels to enhance IFN-γ-mediated PD-L1 expression and sensitizes KRAS-mutant LUAD tumors Fig. 5 Trametinib combined with PD-1/PD-L1 blockade reduces tumor growth in murine KRAS-mutant lung cancer syngeneic models.A Upper panel: Schematic of the experiment.Subcutaneously inoculated KRAS-mutant 393P cells were allowed to growth in syngeneic mice for 9 days.Tumor-bearing mice were randomized in four groups and treated with anti-PD-1 (Anti-PD-1; twice weekly; n = 8 mice per group), trametinib (TRAM; 5 days per week; n = 8 mice per group), their combination (TRAM + anti-PD-1; n = 8 mice per group) or vehicle (control; n = 8 mice per group).Lower panel from left to right: Follow-up of tumor volume over time.Tumor volumes at day 34 of all the experimental groups.Tumor volumes at day 74 of 393P-tumors treated with trametinib alone (TRAM) or in combination with anti-PD-1 (TRAM + anti-PD-1).Kaplan-Meier survival curves for all the experimental groups.B Upper panel: Schematic of the experiment.Subcutaneously inoculated KRAS-mutant LLC cells were allowed to growth in syngeneic mice for 7 days.Tumor-bearing mice were randomized in four groups and treated with anti-PD-1 (Anti-PD-1; twice weekly; n = 6 mice per group), trametinib (TRAM; 5 days per week; n = 6 mice per group), their combination (TRAM + anti-PD-1; n = 6 mice per group) or vehicle (control; n = 6 mice per group).Lower panel from left to right: Follow-up of tumor volume over time.Tumor volumes at day 21 of all the experimental groups.Tumor volumes at day 26 of LLC-tumors treated with trametinib alone (TRAM) or in combination with anti-PD-1 (TRAM + anti-PD-1).Data are expressed as mean ± SD.Comparisons between two experimental groups were performed by two-side t-test; for more than two experimental groups one-way ANOVA followed by the Tukey post hoc test was used to anti-PD-1 treatment.This effect was associated with a less immunosuppressive tumor microenvironment.
Overall, the presence of Id1 in LLC tumor cells impairs the antitumor activity of the combined treatment and restores an immunosuppressive tumor microenvironment.

Discussion
Resistance to the MEK inhibitor trametinib represents a major challenge in the therapeutic management of patients with mutant KRAS LUAD.In this study, we found that trametinib treatment downregulates Id1, a transcriptional regulator involved in the oncogenic mutant KRAS pathway [24], via proteasome-ubiquitin pathway to sensitize KRAS-mutant LUAD tumors to PD-1 blockade immunotherapy.Moreover, our data suggest that Id1 may be involved in the acquired resistance of KRAS-mutant LUAD to trametinib.These data open up new avenues to treating patients with KRAS-mutant lung cancer.
The protumoral phenotype conferred by Id1 expression in KRAS-mutant LUAD has been related to the induction of tumor angiogenesis, tumor-associated immunosuppression and metastasis [19,23,24].Consequently, high levels of Id1 are associated with a reduced overall survival and poor prognosis in these patients [22].This has prompted a series of attempts to pharmacologically target Id1 activity, including the development of antisense molecules [46], peptide aptamers [47] and the development of small-molecules such as AGX51, an Id family antagonist [48,49].However, the lack of a catalytic domain and the nuclear localization of Id proteins, make them a difficult pharmacological target and hampered the approval of these inhibitors.Currently, none of those therapeutic strategies against Id1 has been clinically tested in patients.Our results reveal that the use of trametinib may represent an interesting alternative to directly target Id1.
The upregulation of Id1 mRNA expression levels observed after trametinib treatment suggests that trametinib-mediated Id1 inhibition is associated with a post-translational regulatory process.Although autophagy has been proposed as a mechanism of Id1 degradation in neuroblastoma cells [38], our data indicate that the autophagy inhibitor CQ does not affect the trametinib-mediated Id1 downregulation in KRASmutant LUAD cells.Members of the Id family, like other cell-cycle regulators, are potential substrates of the ubiquitin (Ub)/26 S proteasome system [50,51].Our RNA-seq analysis reveals a significant enrichment of pathways related with proteasome-ubiquitin system in trametinib-treated KRAS-mutant LUAD cells.Importantly, the blockade of the proteasome abrogated Id1 downregulation in trametinib-treated KRAS-mutant LUAD cells.Similarly, in colorectal KRAS-mutant cancer cells, trametinib downregulates Mcl-1 levels in a ubiquitin-proteasome-dependent manner through E3 ligase activation [43].E3 ligases can regulate Id1 degradation through ubiquitination of Id-family members at internal lysine residues and in N-terminal residues [39,21].Interestingly, we found that trametinib-mediated Id1 degradation in KRAS-mutant LUAD cells was associated with the upregulation of the E3 ubiquitin ligases SMURF-2 and FBXW7.Indeed, the inhibition of SMURF2 with a specific shRNA, partially blocked trametinib-mediated Id1 inhibition.Collectively, these data may suggest that trametinib fuels the proteasome activity to degrade Id1 in KRAS-mutant LUAD cells via upregulation of E3 ubiquitin ligases.
There is compelling evidence that some targeted therapies may sensitize tumors to immune-mediated killing, improving ICIs efficacy in NSCLC [17,52].Using preclinical experimental models, we have shown that trametinib and PD-1 blockade combined treatment significantly reduced KRAS-mutant LUAD tumor growth in a synergistic manner.In this regard, the combined treatment increased the proportions of tumor-infiltrating CD8 + T cells and DCs.Moreover, the combined treatment reduced the proportions of the immunosuppressive intratumoral Treg cells, total MDSCs and PMN-MDSCs, whereas the ratio of CD8/Treg was increased.These data indicate that the combined treatment reverses the LLC immunosuppressive microenvironment and promotes a specific antitumor immune response.In concordance with our results, other clinical and preclinical data have demonstrated that MEK1/2 inhibition enhances CD8 + T cells activity and reduces the infiltration of MDSC to sensitize tumors to ICIs [12,53].
Our data indicate that trametinib enhances IFN-γmediated PD-L1 expression on KRAS-mutant LUAD cell surface via Id1 downregulation.Indeed, trametinib/ IFN-γ-mediated PD-L1 expression was abolished when the Id1 levels were restored.Previously, we found that Id1 downregulation enhances IFN-γ-mediated PD-L1 expression on KRAS-mutant LUAD cells [19].High PD-L1 levels in neoplastic tissues have been associated with enhanced objective response rates to PD-1/PD-L1 blockade in NSCLC tumors [54,55].According to our data, an inverse correlation between Id1 and PD-L1 mRNA levels was found in KRAS-mutant LUAD human tumors [19].The synergy of MEKi with anti-PD-1/PD-L1 antibodies has been previously reported in NSCLC [12,56], but none of these studies provide mechanistic evidence for trametinib-mediated sensitization of KRASmutant LUAD human tumors to PD-1/PD-L1 blockade.In this study, we have identified for the first time that this sensitization depends of the trametinib-mediated Id1 inhibition.In this regard, in LLC cells in which exogenous Id1 (Id1-flag) was expressed independent of trametinib treatment, the trametinib-induced PD-L1 expression upon IFN-γ incubation was abolished and importantly, the trametinib and anti-PD-1 combined treatment did not modify the immunosuppressive tumor microenvironment.Collectively, these data indicate that Scale bar: 200 μm.D Flow cytometric quantification of total tumor-infiltrating CD8 + T cells (CD45 + , CD3 + , CD8 + , CD44 + ), Treg cells (CD45 + , CD3 + , CD4 + , CD25 + , Foxp3 + ), total MDSCs (CD45 + , CD11b + , Ly6C + ), PMN-MDSCs (CD45 + , CD11b + , Ly6C + , Ly6G High ).CD8 T cells/Treg ratio is also shown.Data are expressed as the percentage of total leukocytes (CD45 + ), except for Treg cells, which are expressed as the percentage of total CD4 + T cells.Data are expressed as means ± SD.Comparisons between experimental groups were performed by two-sided t-test (parametric) or two-sided Mann-Whitney U-test (non-parametric) (See figure on next page.)trametinib sensitizes KRAS-mutant LUAD tumors to PD-1/PD-L1 blockade via Id1 inhibition.Trametinib has shown efficacy in multiple types of cancer, such as in BRAF-mutant melanoma and NSCLC patients achieving long-term benefit [10,35].However, KRAS-mutant LUADs eventually develop resistance to treatment [9,10].Multiple mechanisms of acquired resistance to MEK inhibition have been reported in different tumor types [57,58].One of the best characterized mechanisms involves the reactivation of the MAPK pathway [59,60].Our study demonstrates that, in contrast to trametinib sensitive KRAS-mutant LUAD, in TR KRAS-mutant LUAD cells, trametinib does not downregulate Id1 levels.Importantly, genetic abrogation of Id1 restored the sensitivity of TR-cells to trametinib suggesting a role for Id1 in the acquired resistance to trametinib in KRAS-mutant LUAD.Functional studies in which KRAS-mutant LUAD are genetically modified to restore Id1 levels support an active role of Id1 in the resistance to trametinib.Moreover, we demonstrate that in TR KRAS-mutant LUAD cells c-Myc and FOSL1 levels, which are well-described mechanisms of targeted therapies against the KRAS pathway [24,43,44], are dependent on Id1 expression levels.These results suggest that in KRAS-mutant LUAD tumors Id1 may sustain the oncogenic KRAS-MAPK signaling pathway through the activation of different kinases or transcription factors.Nevertheless, further investigation is warranted to better understand the role of Id1 in the primary resistance to MEKi.
The possibility of pharmacologically targeting Id1 with trametinib in human cancers could offer a therapeutic alternative for those KRAS-mutant LUAD patients who poorly respond to ICIs or develop early acquired resistance.In this regard, an ongoing phase Ib/II clinical trial (NCT03225664) is currently evaluating the safety, efficacy and maximum tolerated dose of trametinib in combination with pembrolizumab in pretreated NSCLC patients.Moreover, the implication of Id1 gene in trametinib acquired resistance could also serve to design novel immunotherapy combinations, particularly in the context of KRAS-mutant tumors.

Conclusions
In conclusion, we found that trametinib activates the proteasome to reduce Id1 levels in KRAS-mutant LUAD tumor cells.The antitumor effects of trametinib in KRAS-mutant LUAD cells depend on trametinibmediated Id1 downregulation.Moreover, we found that Id1 plays a major role in the acquired resistance to trametinib treatment in KRAS-mutant LUAD tumor cells.Using KRAS-mutant LUAD tumor mouse models we have identified that trametinib-mediated Id1 downregulation sensitizes KRAS-mutant LUAD tumors to anti-PD-1/PD-L1 blockade.Our data suggest a prominent role for Id1 in the antitumor activity of trametinib.

Fig. 1
Fig. 1 Trametinib reduces Id1 protein levels in KRAS-mutant LUAD in vitro and in vivo.A Cell viability assay of KRAS-mutant mouse LUAD cells treated with trametinib (TRAM) 100 nM for 72 h.B Cell viability of KRAS-mutant human LUAD cells treated with trametinib (TRAM) 500 nM for 72 h.C Cell viability of KRAS-wild type human LUAD cells treated with trametinib (TRAM) 500 nM for 72 h.D Western blot analysis of Id1 protein in KRAS-mutant mouse LUAD cells treated as in A. ß-actin was used as control.E Western blot analysis of Id1 in human KRAS-mutant (upper panel) and in human KRAS-wild type LUAD cells (lower panel) treated as in B-C.HSP90 was used as control.F Immunohistochemical quantification of Id1 expression in CMT167 and LLC tumors harvested at day 19 from mice treated with vehicle (Control) or trametinib (TRAM).Left panel: Barr graphs showing the Id1 expression.Right panel: Representative Id1 immunohistochemical stainings.In Western blot analyses, relative optical density is indicated underneath each lane.Data are expressed as mean ± SD.Comparisons between experimental groups were performed by two-sided t-test (parametric) or two-sided Mann-Whitney U-test (non-parametric)

(
Fig S8B)No remarkable differences were observed in the proportions of tumor-infiltrating CD4 + T cells and natural killer (NK) cells.No differences in the expression of the T cell exhaustion marker LAG-3 were found in CD4 + and CD8 + T cells.The exhaustion marker PD-1 was only inhibited in tumor-infiltrating CD4 + and CD8 + T harvested from anti-PD-1-treated tumors (Supplementary Fig.S8B).Tumor-infiltrating myeloid subpopulations were also analyzed.The combined treatment significantly reduced the proportion of total

(Fig. 6
See figure on next page.)Trametinib in combination with PD-1 blockade increases the frequency of tumor-infiltrating effector T cells and reduces the proportion of immunosuppressive Treg cells and MDSCs within the tumor microenvironment.A Left panel: Immunohistochemical quantification of Id1 and tumor-infiltrating CD8 + T cells (CD8), CD4 + T cells (CD4), Treg cells (Foxp3) in LLC tumors harvested from mice shown in Fig. 5B.CD8 T cells/ Treg ratio is also shown.Right panel: Representative images of the immunostainings.Scale bar: 200 μm.B Flow cytometric quantification of tumor-infiltrating CD8 + T cells (CD45 + , CD3